SUMMARY The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an ~ 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and consequently ubiquitylation.

Brain-derived neurotrophic factor (BDNF) is an abundant neurotrophin in brain and peripheral nerves, where it affects neural development, survival and repair after injury. BDNF has been detected in rat and human blood, but the source of circulating BDNF is not established. BDNF messenger and peptide were detected in cultured cells and in the culture medium of human umbilical vein endothelial cells. The expression of BDNF was up-regulated by elevation of intracellular cAMP and down-regulated by Ca 2+ ionophore, bovine brain extract and laminar fluid shear stress. These results suggest that vascular endothelial cells may contribute to circulating BDNF.z 2000 Federation of European Biochemical Societies.

Although several ion channels have been reported to be directly modulated by calcium-calmodulin, they have not been conclusively shown to bind calmodulin, nor are the modulatory mechanisms understood. Study of the olfactory cyclic nucleotide-activated cation channel, which is modulated by calcium-calmodulin, indicates that calcium-calmodulin directly binds to a specific domain on the amino terminus of the channel. This binding reduces the effective affinity of the channel for cyclic nucleotides, apparently by acting on channel gating, which is tightly coupled to ligand binding. The data reveal a control mechanism that resembles those underlying the regulation of enzymes by calmodulin. The results also point to the amino-terminal part of the olfactory channel as an element for gating, which may have general significance in the operation of ion channels with similar overall structures.

RING finger proteins constitute the large majority of ubiquitin ligases (E3s) and function by interacting with ubiquitin-conjugating enzymes (E2s) charged with ubiquitin. How low-affinity RING-E2 interactions result in highly processive substrate ubiquitination is largely unknown. The RING E3, gp78, represents an excellent model to study this process. gp78 includes a high-affinity secondary binding region for its cognate E2, Ube2g2, the G2BR. The G2BR allosterically enhances RING:Ube2g2 binding and ubiquitination. Structural analysis of the RING: Ube2g2:G2BR complex reveals that a G2BR-induced conformational effect at the RING:Ube2g2 interface is necessary for enhanced binding of RING to Ube2g2 or Ube2g2 conjugated to Ub. This conformational effect and a key ternary interaction with conjugated ubiquitin are required for ubiquitin transfer. Moreover, RING:Ube2g2 binding induces a second allosteric effect, disrupting Ube2g2: G2BR contacts, decreasing affinity and facilitating E2 exchange. Thus, gp78 is a ubiquitination machine where multiple E2-binding sites coordinately facilitate processive ubiquitination.

SUMMARY Cue1p is an integral component of yeast endoplasmic reticulum (ER)-associated degradation (ERAD) ubiquitin ligase (E3) complexes. It tethers the ERAD ubiquitin-conjugating enzyme (E2), Ubc7p, to the ER and prevents its degradation, and also activates Ubc7p via unknown mechanisms. We have now determined the crystal structure of the Ubc7p-binding region (U7BR) of Cue1p with Ubc7p. The U7BR is a unique E2-binding domain that includes three α-helices that interact extensively with the ‘backside’ of Ubc7p. Residues essential for E2 binding are also required for activation of Ubc7p and for ERAD. We establish that the U7BR stimulates both RING-independent and dependent ubiquitin transfer from Ubc7p. Moreover, the U7BR enhances ubiquitin-activating enzyme (E1)-mediated charging of Ubc7p with ubiquitin. This is the first example where an essential component of E3 complexes both binds to E2 and enhances E2 loading with ubiquitin. These findings provide new insights into mechanisms of stimulating ubiquitination.

Recognition of ubiquitin and polyubiquitin chains by ubiquitin-binding domains (UBDs) is vital for ubiquitin-mediated signaling pathways. The endoplasmic reticulum resident RING finger ubiquitin ligase (E3) gp78 regulates critical proteins via the ubiquitin-proteasome system to maintain cellular homeostasis and includes a UBD known as the CUE domain, which is essential for function. A probable role of this domain is to recognize ubiquitin modified substrates, enabling gp78 to assemble polyubiquitin chains on these substrates and mark them for degradation. Here, we report the molecular details of the interaction of gp78CUE domain with ubiquitin and diubiquitin. The gp78CUE domain exhibits a well-defined set of interactions with ubiquitin and a dynamic, promiscuous interaction with diubiquitin chains. This leads to a model where the CUE domain functions to both facilitate substrate binding and enables switching between adjacent ubiquitin molecules of a growing chain to facilitate processivity in ubiquitination.

We demonstrate improved accuracy in protein structure determination for large (>/=30 kDa), deuterated proteins (e.g. STAT4(NT)) via the combination of pseudocontact shifts for amide and methyl protons with the available NOEs in methyl-protonated proteins. The improved accuracy is cross validated by Q-factors determined from residual dipolar couplings measured as a result of magnetic susceptibility alignment. The paramagnet is introduced via binding to thiol-reactive EDTA, and multiple sites can be serially engineered to obtain data from alternative orientations of the paramagnetic anisotropic susceptibility tensor. The technique is advantageous for systems where the target protein has strong interactions with known alignment media.

We demonstrate a novel methodology to disrupt the symmetry in the NMR spectra of homodimers. A paramagnetic probe is introduced sub-stoichiometrically to create an asymmetric system with the paramagnetic probe residing on only one monomer within the dimer. This creates sufficient magnetic anisotropy for resolution of symmetry-related overlapped resonances and, consequently, detection of pseudocontact shifts and residual dipolar couplings specific to each monomeric component. These pseudocontact shifts can be readily incorporated into existing structure refinement calculations and enable determination of monomer orientation within the dimeric protein. This methodology can be widely used for solution structure determination of symmetric dimers.